Dynamics of Small Heat Shock Protein Distribution within the ...

Tm JOURNAL OF BIOWGICAL CHEMISTRY

Vol. 269,No. 46,Issue of November 18,pp. 28676-28682, 1994 Printed in U.S.A.

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamics of Small Heat Shock Protein Distribution withinthe Chloroplasts of Higher Plants* (Received for publication, July 5, 1994, and in revised form, September 2, 1994)

Katherine

W.Osteryoung and Elizabeth Vierling

From the Department of Biochemistry, University of Arizona, lhcson, Arizona 85721

Accumulation of the small heat shock proteins (sHSPs) in response to high temperature stress is thought to contribute to the development of thermotolerance in eukaryotic organisms, but the mechanism of action is unknown. We are investigatingthe chloroplastlocalized sHSP, HSP21,with the goal of defining its contribution to the acquisition of thermotolerance in plants. Following an initial heat stress and period of recovery, HSP21 is localized primarily in the soluble fraction of the chloroplast. Duringan additional stress, HSP2l undergoes a temperature-dependent redistribution from the soluble to the insoluble chloroplast fraction in both isolated organelles and intact plants. The change in HSP21 partitioning is accompanied by depletion of the 10-11 S HSP21-containing complexes from the soluble chloroplast fraction. HSP21 in the insoluble fraction cannot be solubilized by nonionic detergent under conditions that release essentially allthe pigments and proteins from the thylakoid membranes,indicating that HSPZl in its insoluble state is not dependent for its insolubility on attachment to an intact membrane. The temperature-dependent redistribution of HSP21 is affected by light intensity but occurs in both leafand root plastids, suggesting that the function of this activity is not strictly related to the presence of the photosynthetic apparatus. Our study indicates that the chloroplast sHSP has dynamic properties similar to those of cytoplasmic sHSPs from plants and other organisms and suggests that the ability to partition between a soluble and an insoluble state reflects a functionally important property of all sHSPs.

with regard to the terminology applied to these structures, they can be categorized as follows based on their sedimentation behavior. The smallest and best-characterized are soluble oligomeric complexes ranging from 200 t o 800 kDa (10-20 S) depending on the organism (8-14). Limited evidence suggests they are composed primarily of sHSPs although they may associate loosely with other macromolecules (9, 11, 15). These complexes appear to be the precursors for formation of two types of larger sHSP-containing particles (8, 16). One type is heterogeneous in size (roughly equivalent to large polysomes) and does not pellet during a very low-speed centrifugation (1,000-15,000 x g).These structures, which have been characterized primarily in plantcells (17-19), have been termed heat also shock granules by Nover (171, although the same term has been applied to the 10-20 S oligomers described above (11)that Nover and colleagues referred to as pre-heat shock granules (16). The composition of these structures has not been well established, but their associationwith specific mRNAs and proteins hasbeen reported (16).A third type of complex consists of extremely large sHSP-containing aggregates (8, 20-23) whose composition is poorly defined. Typically, these structures sediment at very low speeds (1,000 to 15,000 x g ) and are insoluble in nonionic detergent. They are exemplified by the “stress granules” described by Collier and colleagues (20, 21). Formation of both heat shock granules and insoluble aggregates from the 10-20 S oligomers occurs during high temperature treatment and is reversible during recovery in vivo (16, 20, 22). Recently, a fourth molecular form of the mammalian sHSP hasbeen described that derives from disassembly of the 500-kDa form of this protein into a smaller complex of 70 kDa concomitant with its phosphorylation (24). Collectively, these structures constitute a dynamic system in which the deployphysThe small heatshock proteins (sHSPs)’ are anevolutionarily ment of the sHSPschanges with time, temperature, and conserved family of proteins induced by high temperature iological state. Thefunctional implications of this complex sysstress in all eukaryotes. They are encoded in the nucleus and tem remain tobe determined. Interest in the sHSPs hasintensified recently with the disrange in massfrom 15 to 30 kDa. Although diversein size and primary structure, the sHSPs can be identified on the basisof covery that, like the HSPGOs, HSP70s, and HSP9Os (251, the their similar hydropathy profiles and homology to the a-crys- sHSPs and a-crystallins exhibit activities in vitro consistent tallin proteins of vertebrates, which are abundant in the eye with a molecular chaperone function. These activities include lens and arealso heat-inducible in other tissues(1).A number the ability to inhibit aggregation of thermally denatured proteins andto facilitate refolding and reactivation of thermally or of studies have implicatedboth sHSPs and a-crystallins in protection from thermal shock (2-71, but the mechanism of chaotropically denatured enzymes (26-28). While specific in vivo substrates for sHSP activity have notbeen identified, sevprotection is unknown. both sHSPs and a-crystallins interact In vivo, the distribution of sHSP protein in the cytoplasm eral studies suggest that appears t o shift dynamically among several types of sHSP- with components of the cytoskeleton. Co-localization of sHSPs network hasbeen demoncontaining complexes. Although the literature is inconsistent withtheintermediatefilament strated in heat-stressedDrosophila cells (29) and chicken em* This work was supported by National Institutes of Health Grant bryo fibroblasts (20,211. The sHSPfrom turkey co-purified with R01 GM42762 (to E. V.)and by National Institutes of Health Postdoc- actin from smooth muscle tissue and inhibited actinpolymeritoral Fellowship F32 GM14953(to K.W. 0.).The costs of publication of zation in vitro (30, 31). In a bovine lens extract, a-crystallin this article were defrayed in part by the payment of page charges. This could be co-immunoprecipitated withthe intermediate filament article must thereforebe hereby marked “aduertisernent”in accordance protein vimentin usinga monoclonal antibody directed against with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: sHSP, small heat shock proteins;PAGE, vimentin. In the samestudy, a-crystallin wasshown to bind to and promote the disassembly of preformed, insoluble intermepolyacrylamide gel electrophoresis.

28676

Dynamics of HSP21 Distribution in theChloroplast diate filaments in vitro (32). Lavoie et al. (7) reported that constitutive overexpression of Chinese hamster HSP27 in mouse cells partially prevented actin depolymerization by cytochalasin D and reduced cytochalasin-induced growth inhibition. These and other observations of sHSP and a-crystallin interaction with thecytoskeleton have led to the proposal that sHSPs and a-crystallins are involved in chaperoning cytoskeleta1 reorganization and repair in response to heat stress (20, 32). In contrast with other organisms where only one or a few sHSPs are induced by high temperature stress, in plants the heat shock response is dominated by synthesis of the sHSPs. As many as 30 distinct sHSPs have been identified in some plant species (33), suggesting that these proteins critical are for plant survival following exposure to high temperature.Whereas sHSPs have been detected only in the cytosol in other eukaryotes, in plants the sHSPs comprise fourgenefamilies whose encoded proteins arelocalized in threesubcellular compartments: thecytosol, endoplasmic reticulum, andchloroplast (33). The cytosolic sHSPs from plants have properties incommon with those from other eukaryotes. They form oligomeric complexes, undergo reversible transition between small and large complexes (161, and havechaperone-like activityin vitro.' The abundance anddiversity of sHSPs in plantsmay reflect a sessile mode of existence which precludes avoidance of high temperatures often encountered in the natural environment. We are investigating thechloroplast-localized sHSP, HSP21, with the goal of defining its role in the chloroplasts of higher plants during heat stress.HSP2l is unique among the sHSPs in possessing a methionine-rich domain predicted to form an amphipathica-helix (34). This domain is highly conserved across divergent plant species and may reflect a specialized function or substrate specificity within the chloroplast. Like other nuclear-encoded chloroplast proteins, HSP2l is synthesized in thecytoplasm as a large precursor bearing a cleavable N-terminal transit peptide that directs it post-translationally to the chloroplast (35). HSP2l is not detectable in the absence of stress, but is highly induced by elevated temperatures (36, 37) and isfound in a 10-11 S complex in thechloroplast stroma (12, 14) similar t o the soluble oligomeric complexes described above for the cytoplasmic sHSPs. The localization of HSP2l t o an easily isolated organelle provides a distinct advantage for studying the role of sHSPs during heat stress. To investigate whether the chloroplast sHSP exhibits dynamic properties analogous t o those of cytoplasmic sHSPs, we undertook a n analysis of its biochemical behavior during heat stress andrecovery using the pea as a model system. Here we demonstrate that HSP2l partitions between the soluble and insoluble fractions of the chloroplast as a function of temperature both in isolated organelles and in whole plants. Our results establish that the chloroplast sHSP comprises a dynamic, multicomplex system similar t o those described for the cytoplasmic sHSPs. We conclude that theability to undergo a temperature-dependent transition in solubility is a feature comsHSPs and reflects a functionallysignificant mon toall component of their structure orbiological activity in the cell. MATERIALSANDMETHODS Plant Growth and Heat Stress IIFeatments-Pea seeds (Pisum satiuum cv "Little Marvel") wereplanted in vermiculite and grown at 22 to 24 "C at a light level of about 175 pmol m-2 s'l using a day length of 16 h. Plants were watered as needed with one-quarter strength Hoagland's solution. Plants used forchloroplast isolations were harvested 8 to 9 days after planting usually just as thefirst leaves were beginning toexpand. For isolations from heat-stressed plants, plants were placed in a program-

' G. Lee and E. Vierling, manuscript in preparation.

28677

mable growth chamber in the light and subjected to a gradual heat stress regime described previously (36). Briefly, the temperature was increased at a rate of 4 "C h" from 22 t o 38 "C, maintained at 38 "C for 4 h, and then lowered at the same rate. A humidifier was used to prevent transpirational cooling of the leaves. Maximum accumulation of HSP2l occurs under these conditions (36). For analysis of whole tissue, plants were harvested at the times indicated inthe figure legends. Chloroplast Isolation-For all chloroplast isolations, plants were kept in the dark overnight and returned to the light 2 h prior to harvesting. Chloroplasts from heat-stressed plants were isolated 18 h after the end of the interval at 38 "C. Intact chloroplasts were isolated essentially as described previously(38) and resuspended in import buffer containing protease inhibitors (IBP: 50 mM Hepes-KOH, pH 8.0,330 mM sorbitol, 1 mM benzamidine, 5 mM eaminocaproic acid) at a concentration of 3 to 4 mg of chlorophyll m1-I. The chloroplast suspension was kept on ice and used for import reactions within 90 min of chloroplast isolation. Chlorophyll was determined by the method of Arnon (39). In Vitro Import Reactions-An in vitro transcript of pea HSP2l (35) or poly(A)' RNAfrom heat-stressed or unstressed plants was translated in a wheat germ translation system in the presence of [35Slmethionine (ICN)as described previously(40).Import reactions were initiated 45 to 60 min after the translation reactions were started. Imports were carried out in IBP and contained 50 pl of the translation mixture and 200 volume of 300 pl. Somereactions were to 300 pg of chlorophyll in a total scaled down 2-fold.Import was allowed to proceed for 20 min in thelight with gentle shaking. Import reactions were terminated by diluting the reaction mixtures with several milliliters ofcoldIBP. Intact chloroplasts were reisolated as described (38) and resuspended in IBP at a concentration of 1.6 to 1.7 mg of chlorophyll ml-'. Chloroplasts were not treated with protease following import. Heat Deatment and Processing of Isolated ChloroplastsChloroplasts were subjected to heat treatments immediately following import and reisolation or, where imports were not done, immediately after initial isolation of the chloroplasts. When chloroplasts were not used for import, they were first diluted with IBP to a concentration of 1.6 to 1.7 mg of chlorophyll ml-I. Thirty-microliter aliquots of the appropriate chloroplast suspension, equivalent to 50 pg of chlorophyll, were placedin several 1.5-mlmicrocentrifuge tubes on ice. The tubes were cappedand incubated for 10 min in a water bath atthe indicated temperature. After incubation at 43 "C, the highest temperature used in these experiments, greater than 80%of the chloroplasts remained intact based on their ability to pellet through 40% Percoll(38). Following heat treatment, the tubes were returned to ice and 150 pl ofcold lysis buffer (10 m~ Hepes-KOH, pH 8.0, 1 mM benzamidine, 5 mM E-aminocaproic acid, 0.5 mg ml-1 each leupeptin, aprotinin, and antipain) was immediately added. Where indicated, 1.17% TritonX-100 was included in the lysis buffer so that addition of 150 p1 to 50 pg of chlorophyll yielded a detergent:chlorophyllratio of 35:l (w/w).The tubes were vortexed thoroughly to lysethe chloroplasts and centrifuged for 15 min at 14,000rpm in a microcentrifuge at 4 "C. The supernatant was removedto a cold tube, and the pellet was washed in 180 pl of cold lysis buffer by vortexing thoroughly and centrifuging again for 15 min. The wash solution, which contained very little HSP2l when the initial supernatant was completelydrawn off the pellet, was discarded, and the pellet was resuspended in 180 p1of SDS sample buffer. For two-dimensional gel analysis, pellets were resuspended in urea sample buffer (41). Processing of Whole Plants-Pea plants were grownand subjected to a gradual heat stress 8 to 9 days after planting as described above. Plants were then subjected to a second abrupt heat stress at the indicated temperature 24 to 26 h after the end of the interval at 38 "C. Plants showed no visible signs of damage from this treatment after several days. Leaf or root tissue was harvested immediately following the second stress by homogenization in cold sample buffer without SDS or reducing agent at a ratio of 10 mlmg"of tissue using a glass homogenizer. The homogenates were transferred to 1.5-ml tubes and microcentrifuged for 15 min at 4 "C. The pellets were resuspended in the same buffer and centrifuged a second time. Followingthe wash, the pellets were resuspended in the same volume of SDS sample buffer as was used for the initial homogenization. Electrophoretic Analysisof Samples--All samples were analyzed by standard SDS-PAGE on 12.5% polyacrylamide gels. Except whereindicated, volumes loaded on the gels represent equivalent proportions of the pellet and soluble fractions from each sample. Some soluble fractions were also analyzed by nondenaturing pore exclusion gel electrophoresis (14, 42), using a 4 to 20% gradient of polyacrylamide, or by standard two-dimensional gel electrophoresis (41).For radiolabeled

28678

Dynamics of HSP21 Distribution in the Chloroplast

samples, gels werefixed, infiltrated with 1 M sodium salicylate in30% methanol, dried, and exposed to film. In some experiments, the radioactivity inspecific protein bands was quantifiedby scintillation counting following excision of the bands from driedgels.Forunlabeled samples, gels were blotted to nitrocellulose and HSP2l was detected by immunoblotting usinga 1:3000dilution of crude anti-HSP2l antiserum (43). HSP18.1 (44) was detected using a 1:lOOO dilution of crude antiserum. Antigen-antibody complexes were visualized using a chemiluminescent detection system (Amersham). When quantification of immunoblotswasrequired,antigen-antibody complexes weredetected with '"I-protein A (Amersham), and immunoreactive bands were quantified by analysis on a PhosphorImager (Molecular Dynamics).

A

Soluble Pellet Ttl 22 37 40 43

pHSP21 b HSP2l b

r1 2 3 4 5

B

6 7 8 9

Soluble Pellet 22 37 40 43 HSP2l b

RESULTS

22 37 40 43 "C

22 37 40 43 "C

e -0 1 2 3 4

5 6 7 8

Temperature-dependent Redistribution of HSP21 in Isolated Chloroplasts-To investigate the distributionof HSP21 in the chloroplast as a function of temperature, we took advantage of C Soluble Pellet a well characterized in vitro import system employing chloro22 37 40 43 22 37 40 43 "C plasts isolated from young pea plants (40) that had been heatstressed the day before the isolation. An in vitro transcript 74.7 b encoding the pea HSP21 precursor was translated in the presence of ["'Slmethionine t o radiolabel the protein. The transla46.3 b tionproduct was added to chloroplastsisolated from heatstressed pea plants underconditions allowing for import of the 27.6 b precursor and cleavage of its transit peptide. Following the import reaction, chloroplasts were washedand incubatedfor 10 18.6 b min a t 22 "C (control) or a t elevated temperatures rangingfrom 15.8 b 37 to 43 "C. The latter temperatures represent a physiologi1 2 35 46 7 8 cally relevant range within which thermotolerance develops in FIG.1. Temperature-dependent partitioning of HSP2l in chlomany plant species (45, 46). After heat treatment, the chloro- roplasts isolated from heat-stressed pea plants. Plants were subplasts were osmotically lysed and separated into soluble and jected to a gradual heat stress and chloroplasts were isolated after 18 h insoluble fractions by microcentrifugation. The fractions were of recovery. A, partitioning of newly imported HSP21. Radiolabeled translatedin vitro was imported into isolated chloroplasts.Folthen analyzed by SDS-PAGE and fluorography. In chloroplasts HSP2l lowing import and washing, whole chloroplasts were lysed directly in incubated at 22 "C following import, most of the radiolabeled SDS sample buffer (lane I ) or were incubated for 10 min at the indiHSP21 was in thesoluble fraction (Fig. L4, lunes 2 and 6). As cated temperature, lysed, and divided into pellet(lanes2-51 and soluble the temperatureof incubation increased, the amount of radio- (lanes 6-9)fractions. Fractions were analyzed by SDS-PAGE and fluolabeled HSP2l in the pellet fraction increased with a corre- rography. pHSP21, unimported HSP2l precursor that remainsbound to the envelope membranesafterwashing; HSP21, mature,imported sponding decrease in the soluble fraction (Fig. lA, lanes 3-5 HSP2l from which the transitpeptide has been cleaved. B, partitioning and 7-9 1. Although the proportion of HSP2l in thepellet frac- of endogenous HSP21. Immediately following isolation, chloroplasts tion variedfrom experiment toexperiment, a t 43 "C i t normally were incubated for 10 min at the indicated temperature, lysed, and divided into pellet (lanes 1-41 and soluble (lanes 5-8) fractions by ranged from 40-60%. microcentrifugation.Fractionswereanalyzed by SDS-PAGE, and The temperature-dependent redistribution of HSP2l into the HSP2l was visualizedby immunoblotting and chemiluminescent detecinsoluble chloroplast fraction was not restricted to newly im- tion. C , Coomassie-stained gel of extracts from panel A. Sizes of molecported protein. In chloroplasts not used for import of radiola- ular mass markers are indicatedon the left in kilodaltons. beled protein, endogenous HSP2l thataccumulated during the heat stress treatment showed the same temperature-depend- poly(A)+RNA isolated from heat-stressed pea plants were ent change in partitioningas did newly imported protein based added to chloroplasts isolated from heat-stressed plants to efon immunoblot assays (Fig. 1B). The proportion of the total fect import of nuclear-encoded chloroplast proteins. The chloendogenous HSP2l in the pellet fraction at 43 "C was also roplasts werewashed,incubated a t different temperatures, 4040%. Therefore, with regard to their behavior in isolated and divided into soluble and pellet fractions by microcentrifuchloroplasts, both newly imported and pre-existing HSP2l rep- gation. The fractions were analyzedby SDS-PAGEand fluorogresent similar pools of protein. raphy. HSP2l and several other proteins were imported into Examination of stained gels indicatedthat the redistribution the chloroplasts (Fig. 2 A , lane 1). When the chloroplasts were of HSP2l was not the result of bulk thermal aggregation of heated, HSP21increased in thepellet fractionand decreased in chloroplast proteins.Although a temperature-dependent loss of the soluble fraction as a function of temperature (Fig. 2 A , lanes HSP2l from the soluble fraction was observed by immunoblot- 2-9) as wasobserved when HSP21 was the sole import product. ting (HSP21 is not sufficiently abundant to be detected by With the exception of one other polypeptide of about 41 kDa either Coomassie or silver staining), there was little change in (Fig. 2 A , asterisk), there was littleeffect of heat treatment on the staining pattern in either thesoluble or insoluble fraction the solubility of any of the other imported proteins. These rebetween 22 and 43"C (Fig. E ) . These results suggest that the sults indicate that the change in HSP2l solubility is not the temperature-dependent transition to aninsoluble state is spe- result of general proteinaggregation in the heated chlorocific to HSP21. plasts. As stated above, a 41-kDa protein also exhibited temperaSpecificity of Temperature-dependent Redistribution-To investigate further whether theeffect of heat treatment on pro- ture-dependent depletion from the soluble chloroplast fraction tein partitioningbetween the soluble and insoluble chloroplast in a manner similar to that of HSP2l (Fig. 2 A , asterisk). We fractions was specific to HSP21, we examined in more detail have not determined the identityof this protein, but have esthe effects of similar treatments on the distribution of other tablished that it is neither the chloroplast fructose-1,6-bisphoschloroplastproteins. Radiolabeled translation products of phatase, which has a similar mass(47), nora dimerized form of

Dynamics of HSP21 Distribution in Chloroplast the

A

Soluble

Soluble Pellet Ttl 22 37 40 43

28679

22 3740 43 "C

22 37 40 43°C

-?w*

4 74.7

*

4 46.3 4 27.6

LHC D

'1

HSP2l D 4 18.6 4 15.8

SSU b 1

B

2 3 4 5

6 7 8 9

Soluble Pellet Ttl

22374043

22 3740 43 "C 4 74.7

*

4 46.3

-

4 27.6

LHC b

g!

A ssu, w

---

-

415.8

HSPPl b

i

4 42

1 2 3 4 FIG.3. Temperature-dependentredistribution of HSP2l is accompanied by loss of native complexes from the soluble chloroplast fraction. Soluble fractions(lanes 1-4) identical with those shown in Fig. IA, lanes 2-5, were analyzed by nondenaturing pore exclusion gel electrophoresis and fluorography. Sizes of native, soluble forms of HSP2l areindicated on the right in kilodaltons. The arrow is explained in the text.

lieved to be a dimer of HSP21( 1413To determine which of these complexes redistributes into the pellet fraction, isolated chloroplasts were used for import of radiolabeled HSP21 and heattreated as described above. The soluble fractions were analyzed by nondenaturing pore exclusion gel electrophoresis (14, 42) and fluorography. All three forms of HSP2l were reduced in the soluble fraction as the temperature of incubation increased (Fig. 3). While there was a partial loss of dimeric HSP21 with increasingtemperature, a t 43 "C little or no radiolabeled HSP2l remained in the200- and 230-kDa complexes. In addition to the temperature-dependentincrease in insoluble HSP2l described above, there wasalso an increase in the amount of radiolabeled HSP2l present in soluble aggregates that were too large to migrate into thenondenaturing gel (Fig. 3, arrow). These resultsindicate that following high temperaHSP2l which is often detected even under denaturing condi- ture treatment HSP2l becomes associated both with larger soluble particles and with insoluble complexes. tions (14) (resultsnot shown). To determinewhetherthe Assembly ofHSP.21 into HighMolecular Weight Complexes Is change in solubility of the 41-kDa protein was linked with or Not Required for Redistribution-To determine if assembly of dependent upon the change in HSP2l solubility, an import experimentwas conducted usingtranslation products of HSP2l into the high molecular weight complexes is required insoluble chloroplast fraction, the poly(A)+ RNAfrom unstressed plants incombination with chlo- for its redistribution into the roplasts isolated from unstressed plants. In this experiment, radiolabeled HSP21 precursor was imported into chloroplasts isolated from unstressed plants. Temperature-dependent parHSP21, which is strictly heat-inducible (361, was not represented in either thepoly(A)+ RNAor the chloroplasts because titioning of radiolabeled HSP21 into theinsoluble fraction was they were both derived from unstressed tissue. Following im- still observed (Fig. 4). Because no assembled HSP21 import port and heat treatment of the chloroplasts, the 41-kDa protein product is detectable when import is carried out in control was stilldiminished from the soluble fraction as theincubation chloroplasts (141, these results suggest that the transition to temperature increased (Fig. 2B, asterisk). These results indi- the insoluble state does not require that theprotein assemble cate that the change in solubility of this polypeptide occurs into the200- and 230-kDa complexes. However, we cannot rule independently of HSP21. However, since this protein is present out transient assembly prior to insolubilization in this assay. not a result of in both control and heat-stressed tissue,we cannot rule out the These resultsalso suggest that the transition is possibility that the change in its solubility is necessary for thermal self-aggregation of HSP2l because it occurs even in control chloroplasts where the concentration of the protein is redistribution of HSP2l into theinsoluble fraction. The Increase in Pelletable HSP21 Is Accompanied by a extremely low, i.e. when only radiochemical and not immunologically detectable quantities of HSP21 are present. Decrease in Native HSP21 Complexes from theChloroplast HSP21 in the Pellet Fraction Is Not a n Intrinsic Membrane Stroma-The results described above indicate that in chloroplasts isolated 18 h after the end of a 38 "C heat stress,most of Protein-To determine whether HSP2l insolubilityin heatthe HSP2l is localized in thesoluble fraction of the chloroplast treated chloroplasts might be the result of membrane attach(Fig. 1 B , lanes 1and 5).Previous work from our laboratory has ment, we examined the solubility of pelletable HSP2l in Triton from heat-stressed pea shown that this soluble protein can be resolved as two high X-100. Chloroplastswereisolated molecular mass HSP2l-containingcomplexes of approximately 200 and 230 kDa (10-11 S) and a smaller 42-kDa protein beT. Suzuki and E. Vierling, unpublished data.

1 2 3 4 5 6 7 8 9 FIG.2. Specificity of temperature-dependentredistribution of HSP21 in isolated chloroplasts. I n vitro translationproducts of poly(AY RNA isolated from heat-stressed (A) or unstressed ( B ) pea plants were mixed with chloroplasts isolated from heat-stressed (A) or unstressed ( B )plants under conditions allowing for import of chloroplast proteins. Following import and washing,whole chloroplasts were lysed directly in SDS sample buffer (lanes 1 ) or were incubated for 10 min at theindicated temperature, lysed, and divided into pellet (lanes 2-5)and soluble(lanes 6-9)fractions. Fractions were analyzed by SDSPAGE and fluorography. In panel A, 5 times moreof each pellet fraction was loaded on the gel than of the corresponding soluble fraction.Positions of the light-harvestingchlorophyll a/b protein (LHC), HSP21, and smallsubunit of ribulose-1,5-bisphosphate carboxylase ( S S U ) are shown on the left. Sizes of molecular mass markers are indicated on the right in kilodaltons. Theasterisk ( 9 is explained in the text.


28680

Pellet Ttl pHSP21

- -

HSP2l

0

22 37 4043 0

--

Pellet

Soluble 22 37 40 43

Soluble

"

-Trit +Trit -Trit +Trit 22 43 22 43 22 43 22 43 "C

oc

--

1 2 3 4 5 6 7 8 9 FIG.4. Temperature-dependent redistribution of HSP21 occurs in chloroplasts from unstressed plants. Radiolabeled HSP2l translated in vitro was imported into chloroplasts isolated from unstressed plants.Following import and washing, whole chloroplasts were lysed directly in SDS sample buffer (lane 1 ) or were incubated for 10 min at the indicated temperature, lysed, and divided into pellet (lanes 2-5) and soluble(lanes 6-9) fractions. Fractions were analyzedby SDSPAGE and fluorography. pHSP21, unimported HSP2l precursor that HSP21, maremains bound to the envelope membranes after washing; ture, imported HSP2l from which the transit peptide has been cleaved.

-"--

" "

HSP2lb

0

1

2

3

4

5

6

7

8

FIG.5. Pelletable HSP2l is not solubilized by detergent treatment. Chloroplasts isolated from heat-stressed plants were incubated for 10 min a t 22 or 43 "C a s indicated and lysed in either the absence (lanes 1-2 and 5-6) or presence (lanes 3 4 and 7-8) of Triton X-100 using a detergentchlorophyl1 ratioof 35:l. HSP2l in pellet(lanes 1 4 ) and soluble (lanes 5-8) fractions was analyzed by SDS-PAGE and immunoblotting using chemiluminescent detection.

in leaf tissue waslocalized in thesoluble fraction (Fig. 6 A , lanes 1 and 5 ) .Following the second stress, HSP2l redistributed into the insolublefraction in a temperature-dependentmanner plants after18 h of recovery and incubatedat 22 or 43"C for 10 (lanes 2 4 and 6-8) as had been observed in isolated chloroplasts. Extraction of the pellet fraction with 1% or 2% Triton min. The chloroplastswereimmediatelylysed in the same buffer used for the previously described experiments, but con- X-100 did not affect the solubility of pelletable HSP2l (not taining Triton X-100 a t a concentration to yield a detergent shown). Asimilar redistribution of HSP21 also occurred in rechlorophyll ratio of 35:l. This ratio wasshown by Morrissey et stressed root tissue (Fig. 6B ), indicating that the transition to al. (48) tobe sufficient for complete release of chlorophyll and insolubility isnot specific to photosyntheticplastids.Pea the photosystem I1 reaction center from the grana thylakoids, HSP18.1, a class I cytosolic sHSP (44), redistributed in vivo in which are themost detergent-resistant membranes in the chlo- a manner similar to HSP2l (Fig. 6C). These results suggest that the ability undergo to temperature-dependent partitioning roplast. Following microcentrifugation andwashinginthe same buffer, only a small white pelletremained, indicating that between a soluble and an insoluble state is a general property of plant sHSPs. the membranes had been thoroughly disrupted and all the HSP21 Redistribution Is Not Prevented by High Lightchlorophyll solubilized. Coomassie staining of the Triton-extracted pelletfraction following SDS-PAGE confirmed that Glaczinski and Kloppstech (23) reported that association of nearly all the membrane protein had been solubilized (not imported, radiolabeled HSP2l with the insoluble chloroplast shown). Immunoblotanalysis of the pellet and soluble fractions fraction in isolated pea chloroplasts could be inhibited if the demonstrated that HSP2l remained insoluble in the 43"C pel- plants were heat-shocked in high light prior to chloroplast isolation. To address theeffect of light intensityon partitioning of let following detergent treatment (Fig. 5). been heat-stressed Soluble and Pelletable HSP21 Are Not Differentially HSP21 i n vivo, we subjected plants that had Modified-The foregoing experiment indicated that the tem- at 38 "C and allowed to recover for 24 h to a second 43 "C heat perature-dependent change in HSP2l solubility was not the stress at two light intensities equivalent to about one-third or result of a post-translational modification, such as prenylation, one-tenth of full sunlight. Redistribution of HSP21 into the insoluble fraction,as determined by immunoblotting, wasmore that anchors the protein to the membrane. Neither was the lower light level (Fig. 7). solubility change accompanied by a modification that signifi- pronounced at the higher than at the cantly altered the mass of monomeric HSP2l as indicated by DISCUSSION SDS-PAGE analysis of soluble and pellet fractions (Figs. lA, In a previous study from our laboratory, we were unable to 1 B , and 2 A ) . To investigate whether othermodifications might be associated with HSP2l redistribution,we analyzed the mi- detect significant quantities of the chloroplast sHSP in aningration of soluble and pelletable HSP21 by two-dimensional gel soluble fraction of the chloroplast (36). This was in contrast electrophoresis (41). Both forms of the protein migrated iden- with other studiesindicating that cytoplasmic sHSPs undergo tically (not shown), indicating they do not differ detectably in reversible transition between a soluble and an insoluble state charge. These results suggest that HSP2l redistribution is not i n vivo (20,22). It is now evident that theproportion of HSP2l accompanied by a change in the phosphorylation state of the that is insoluble is a function of the time elapsed since impoprotein or by other modifications that would significantly alter sition of the stressas well as of the severity of the stress.If the tissue is sampled 18 to 20 h after recovery from stress, as was its charge. insoluble HSP2l is HSP21 Redistribution Occurs i n Vivo-All the experiments the case in our earlier study (36), little described above were conducted using an isolated chloroplast detected. This isalso true for the cytoplasmic protein HSP18.1. sampled only 2-4 h into the initial heat system. To determine whether HSP2l exhibits similar behavior However, if the tissue is stress, significant amounts of both HSP2l and HSP18.1 can i n vivo, we examined HSP2l partitioning in intact plants. Young pea plants were given an initial heat stressat 38 "C to often be detected in the pellet fraction (results not shown), effect accumulation of HSP21. After 24 to 26 h of recovery, the although theproportions vary between experiments. Additionplants were restressed abruptlyfor 30 mina t various tempera- ally, if plants are restressed aftera period of recovery, HSP2l tures. Based on numerous observations, it isunlikely that sig- and HSP18.1 again become insoluble in proportion to tempernificant accumulationof newly synthesized HSP21 would occur ature. However, a t no time did we observe complete loss of during the second stress under these conditions. Immediately HSP2l from the soluble chloroplast fraction as did Glaczinski following the second stress, leaf or root tissue was harvested by and Kloppstech (23). Although we have not specifically adhomogenizing in a nondenaturing buffer and dividing the ho- dressed the question of the reversibility of HSP21 insolubilizamogenate into pellet and soluble fractions by microcentrifugation, our in vivo results are consistent with previous observation. Samples were analyzed by SDS-PAGE and immunoblot- tions showing that the partitioningof sHSPs intoa detergentting. Atthe endof the recovery period, nearly all of the HSP2l insoluble fraction is reversible in vivo (20,221. Taken together,


A

Pellet

Soluble

28681

the insoluble chloroplast fraction. To explain this result they proposed that binding to the insoluble fraction of endogenous Restress Restress HSP2l synthesized during the heat stress became saturated in 22 38 41 44 22 38 41 44 "C high light, preventing further binding of newly imported protein in isolated organelles. Our results demonstrating greater HSP2l b redistribution of endogenous HSP21 a t higher light intensities in vivo are consistent with this explanation. However, while 1 2 3 4 5 6 7 8 light level may potentiate HSP21 partitioning in leaf plastids, the redistribution behavior of HSP2l is not related solely to the presence of the photosynthetic apparatus because it also occurs Pt Sol in root plastids. 22 41 22 41 "C A concern throughout the course of this investigation has been the possibility that theheat-induced transition of HSP2l to an insoluble state isnot a functional property of the protein but instead represents irreversible aggregation of exposed hydrophobic surfaces following thermaldenaturation. Indeed, most of the published data describing the dynamic properties of Pt Sol sHSPs are consistent with such a mechanism of insolubilizaC tion, and it is surprising that this possibility has received so 22 41 22 41 "C little attention. It is therefore worthwhile summarizing the HSP18.1 b 0 . 0 reasons that thisis probably not the case either for HSP21 or for other sHSPs. 1)The heat-induced insolubilization observed 1 2 3 4 FIG. 6. Redistribution of HSP2l occurs in vivo. Whole pea plants in isolated chloroplasts was specific to HSP21 and only one were given a gradual heat stress to 38 "C and allowed to recover a t other polypeptide. A similar specificity for heat-induced redis22 "Cfor 24 h. Plants were then given an additional 30-min stress a t the tribution has been documented for the mammalian sHSP and indicated temperature. Leaf (A) or root ( B and C) tissue was homog- for HSP70 (8,49). It seems unlikely that HSP2l or other heatenized, and the distribution of HSP2l (A and B ) or HSP18.1 (C) between the pellet ( P t ) and soluble ( S o l ) fractions was determined by inducible HSPs would be more susceptible to thermal denaturSDS-PAGE and immunoblotting using chemiluminescent detection. ation than other proteins in the cell. 2) The observations that Lunes labeled 22 "C show the distribution of HSP2l or HSP18.1 a t the heat-induced sHSP insolubilization is reversible in vivo after a end of the 24-h recovery period. few hours of recovery (8, 20,22) and thatreversibility is correlated with the acquisition of thermotolerance (8,221are inconsistent with the notion that sHSP insolubilization is theresult Pellet Soluble " of nonproductive thermal aggregation. 3) In ourstudy, HSP2l Restr Restr insolubilization was only observed in intact chloroplasts and 22" Hi Lo 22" Hi Lo whole plants. We were unable to detectheat-induced aggregation in a chloroplast lysate or following in vitrotranslation in a wheat germ extract or rabbit reticulocyte lysate (results not HSP21 b shown). Moreover, even though insoluble HSP18.1 was deHSP18.1 purified after overexpression in tected invivo, 1 2 3 4 5 6 FIG.7. Effect of light level on HSP2l redistribution in vivo. Escherichia coli does not undergo thermal aggregation in vitro Whole pea plants were given a gradual heat stressto 38 "C and allowed a t temperatures ashigh as 65 0C.4Neither do heated preparato recover at 22 "C for 24 h (lunes 1 and 4 ). Plants were then given an tions of purified mouse or human sHSPs aggregate in vitro(27, additional 30-min stress a t 44 "C (Restr) a t a light intensity of either 675 pmol m-* s-l (Hi, lunes 2 and 5 ) or 75 pmol m-2 s" (Lo, lunes 3 and 28). These observations suggest that sHSPs do not in general self-aggregate a t high temperatures. However, this argument 6).Leaf tissue was homogenized and thedistribution of HSP2l between the Pellet and Soluble fractions was determined by SDS-PAGE and should be considered with caution because thermal aggregation immunoblotting using chemiluminescent detection. of other proteins has been shown to occur more readily in vivo than in vitro (50). 4) Following import of HSP2l into chloroour results indicate that HSP2l behaves similarly tocytoplas- plasts isolated from recently heat-stressed plants, HSP2l could mic sHSPs with respect to the dynamics of its distribution in be detected in an insoluble form even though the imported the chloroplast. protein had not itself been subjected to high temperature (23). We have demonstrated that the temperature-dependent re- This suggests that HSP2l insolubility reflects a heat-induced distribution of HSP2l is accompanied by a loss of the native change in the physiological status of the chloroplasts. 5 ) HSP2l 200- and 230-kDa complexes from the soluble fraction of the redistribution in vivoduring asecond heat stressafter aperiod chloroplast. Our previous work has shown that these are the of recovery is affected by light intensity. Light intensity by itself predominant soluble forms of HSP2l detectable inheatshould not have an effect on protein denaturation, again sugstressed pea plants (14). These findings suggest that these gesting that theredistribution reflects a change in the physiocomplexes constitute the "active" form of the protein with re- logical state of the chloroplast. These and other observations The tran- support the supposition that the insolubilization behavior of gard to its ability to redistribute during heat stress. sition to the insoluble state does not appear to reflect a post- HSP21 and othersHSPs is not a result of nonproductive thertranslational modification that affects either the charge or mal denaturation of these proteins, but reflects a functionally mass of monomeric HSP21, although it could result from modi- significant aspect of their structure or biological activity. fication of another chloroplast component with which HSP2l Although the heat-induced redistribution of HSP2l is probinteracts. ably not the result of nonfunctional aggregation, the natureof In studying the effect of light on the distribution of HSP21, the structural change that renders the protein insoluble reGlaczinski and Kloppstech (23)found that high lighttreatment mains obscure, as is true for the cytoplasmic sHSPs. It may of plants during heat stress prior to chloroplast isolation inhibited association of newly imported, radiolabeled HSP21 with G. Lee and E. Vierling, unpublished data. "

"

"

-

- -

28682

Dynamics of HSP21 Distribution in

the Chloroplast

reflect aggregation of the soluble 200- and 230-kDa complexes 11. Bentley, N. J., Fitch, I. T., and Tuite, M. F. (1992)Yeast 8, 95-106 12. Clarke, A. K., and Critchley, C. (1992)Plant Physiol. 100, 2081-2089 into largerfunctional aggregates or bindingof these complexes 13. Helm, K. W., LaFayette, P. R., Nagao, R. T., Key, J. L., and Vierling, E.(1993) to an insoluble component of the chloroplast or a component Mol. Cell. Biol. 13, 238-247 that becomes insoluble upon heat stress. Anotherpossibility is 14. Chen, Q., Osteryoung, K., and Vierling E. (1994)J. Biol. Chem. 269, 1321613223 that theprotein becomes membrane-bound. Adamskaand 15. Miron, T., Wilchek, M., and Geiger, B.(1988)Eur. J. Biochem. 178, 543-553 Kloppstech (51) reported that insoluble HSP2l behaves as an 16. Nover, L., Scharf, K.-D., and Neumann,D. (1989)Mol. Cell. Biol.9,1298-1308 intrinsic membrane protein andis associated specifically with 17. Nover, L., Scharf, K.-D., and Neumann,D. (1983)Mol. Cell. Biol. 3,1648-1655 18. L., and Scharf, K.-D. (1984)Eur. J. Biochem. 139, 303-313 the grana thylakoids. They postulated that HSP21, by binding 19. Nover, Mansfield, M. A., and Key, J. L. (1988)Plant Physiol. 86, 1240- 1246 to the thylakoids, exertsa protective effect on photosystem I1 20. Collier, N. C., and Schlesinger, M. J. (1986)J. Cell B i d . 103, 1495-1507 function during heat stress. However, our results demonstrat- 21. Collier, N. C., Heuser, J.,Levy, M.A,, and Schlesinger, M. J. (1988)J. Cell Biol. 106, 1131-1139 ing that pelletable HSP2l remains insoluble under conditions 22. Anigo,A.-P., Suhan,J. P., and Welch, W. J. (1988)Mol. Cell. Biol. 8,5059-5071 that solubilize essentiallyallthemembrane components 23. Glaczinski, H., and Kloppstech, K. (1988)Eur: J. Biochem. 173,579-583 strongly suggest that HSP2l not is dependent for its insolubil- 24. Kato, K., Hasegawa, K., Goto, S., and Inaguma, Y.(1994)J. Biol. Chem. 289, 11274-11278 ity on attachment to the membrane and isvery likely not an 25. Becker, J., and Craig, E. A. (1994)Eur. J. Biochem. 219, 11-23 intrinsic membrane protein. Furthermore, HSP2l also parti- 26. Horwitz, J. (1992)Proc. Natl. Acad. Sei. U. 5. A. 89, 10449-10453 K. B., Groenen, P. J. T. A,, Voorter, C. E. M., de Haard-Hoekman,W. A., tions into the pellet fraction in nonphotosynthetic root plastids, 27. Merck, Horwitz, J., Bloemendal, H., and de Jong,W.W. (1993)J . B i d . Chem. 268, which do not contain thylakoid membranes. Therefore, it is 1046-1052 likely that thefunction of HSP2l redistribution in the plastids 28. Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993)J. B i d . Chem. 268, 1517-1520 of plant cells entails protection of some process or structural 29. Leicht, B. G., Biessmann, H., Palter,K. B., and Bonner, J. J. (1986)Proc. Natl. feature common to both leaf and root plastids. Acad. Sei. U. S. A. 83, 90-94 The physiological significance of HSP2l dynamics in lightof 30. Miron, T.,Wilchek, M., and Geiger, B. (1988)Eur. J. Biochem. 178,543-553 31. Miron, T.,Vancompemolle, K., Vandekerckhove, J., Wilchek, M., and Geiger, recent evidence for sHSP and a-crystallin chaperone-like activB.(1991)J. Cell Biol. 114, 255-261 ity and interactionwith the cytoskeleton is unclear. The simi- 32. Nicholl, I. D., and Quinlan, R. A. (1994)EMBO J. IS, 945-953 larity in behavior between HSP2l and other sHSPs could re- 33. Vierling, E. (1991)Annu. Rev. Plant Physiol. Plant Mol. Biol.42, 579-620 34. Chen, Q., and Vierling, E. (1991)Mol. & Gen. Genet. 226,425-431 flect a common function in protection or repair of large, 35. Vierling, E., Nagao, R. T., DeRocher, A. E., and Harris, L. M. (1988)EMBO J. 7,575-581 insoluble complexes similar t o the cytoskeleton in response to Q., Lauzon, L. M., DeRocher, A. E., and Vierling, E. (1990)J . Cell Biol. heat stress.Although cytoskeletal elements havenot beeniden- 36. Chen, 110,1873-1883 tified inhigherplant chloroplasts,“microtubule-like struc- 37. Osteryoung, K.W., Sundberg, H., and Vierling, E. (1993)Mol. & Gen. Genet. 239,323-333 tures” have been described (52-54). An important step in elu38. Mishkind, M. L., Wessler, S. R., and Schmidt, G.W. (1985)J. Cell Biol. 100, cidating thefunction of the dynamicbehavior of sHSPs will be 226-234 determining the composition of the insoluble complexes with 39. Amon, D. I. (1949)Plant Physiol. 24, 1-15 40. Vierling, E., Mishkind, M. L., Schmidt, G. W., and Key, J. L. (1986)Proc. Natl. which they associate. Acad. Sci. U.5. A. 83, 361-365 41. OFarrell, P. H. (1975)J. Biol. Chem. 250,

4007-4021

Acknowledgments-We aregrateful to Dr. GarrettLeeandTen 42. Andersson, L.-O., Borg, H., and Mikaelsson,M. (1972)FEBSLett. 20,199-202 Suzuki for thoughtful critiques of the manuscript and to Dr. J. Lopez 43. Vierling, E., Harris, L. M., and Chen, Q. (1989)Mol. Cell. B i d . 9,461-468 Gorge for generously providing antibodies against the chloroplast fruc-44. DeRocher, A. E., Helm, K. W., Lauzon, L.M., and Vierling, E. (1991)Plant Physiol. 96, 1038-1047 tose-1,6-bisphosphatase. REFERENCES

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